During or after translation, many eukaryotic proteins are further modified in vivo by an enzymatic process, namely, glycosylation, which attaches glycans to the proteins. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. For reviews on protein glycosylation, see, for example, Lis and Sharon, Eur. J. Biochem. 218:1-27 (1993). One exemplary function of glycosylation is to increase the solubility of the protein substrate. Glycosylation is often species- and cell-specific, and is determined as well by the structure of the protein backbone and the carbohydrate attachment site. However, during protein expression in eukaryotic cells containing glycosylation machineries, different product species from a single protein template are often inevitably produced, containing various lengths or components of glycan side chains. Thus, there is a need to control glycosylation during protein expression to achieve a product of improved purity. Among well-known technologies, site-directed mutagenesis is often used to remove the glycosylation site of the protein to be expressed, resulting in protein products with less or no glycosylation at the site.
The murine anti-factor B antibody produced by the hybridoma clone 1379 (mAb 1379) and its humaneered antibodies or fragments are disclosed in U.S. Patent Publication No. US 2005/0260198 A1, now U.S. Pat. No. 7,999,082 and U.S. Patent Publication No. US 2008/0299114, now U.S. Pat. No. 7,964,705, which are incorporated herein by reference in their entirety. One of these humaneered anti-factor B antigen-binding fragments, TA106, contains a consensus triplet amino acid residue sequence, which represents thematically a potential glycosylation site, in its light chain CDR1 domain. Thus, for future eukaryotic expression and purification of TA106 or TA106-related antibodies or fragments, there is a need to determine whether the expressed antibodies or fragments are actually glycosylated at this site. If the expressed antibodies or fragments are actually glycosylated at this site, there is a further need to reduce or eliminate the site-specific glycosylation to improve product homogeneity, while at the same time maintaining the binding characteristics of such anti-factor B antibodies or fragments, e.g., the binding specificity, the binding efficacy, etc.
All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.